Riboswitch modulated gene therapy for retinal diseases

ABSTRACT

The present invention provides constructs comprising modified riboswitches to regulate expression of a transgene within a subject. Methods of treating a disease, specifically an eye disease, are also contemplated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims priority to U.S. Provisional Application No.62/469,705 filed on Mar. 10, 2017, the contents of which areincorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

N/A

BACKGROUND OF THE INVENTION

Age-Related Macular Degeneration (AMD) and glaucoma are leading causesof vision loss worldwide. AMD is a common eye disease among people age50 and older. In AMD, there is damage to the macula, a small area madeup of millions of light-sensing cells near the center of the retina andthe part of the eye needed for sharp, central vision, and the ability tosee objects that are straight ahead. Macular damage is caused by theformation of deposits called drusen, and in some cases, the growth ofabnormal blood vessels, under the retina.

Glaucoma is a group of diseases in which the eye's optic nerve isdamaged resulting in vision loss and blindness. The hallmark of glaucomais increased intraocular pressure related to build-up of fluid (aqueoushumor). It some patients the disease is genetic. In other patients,inflammation in the eye is thought to be involved in glaucoma.

Neither AMD nor glaucoma can be prevented. There is no treatment forearly AMD in which there is no symptoms or loss of vision. Advanced AMDis treated with biologics. Glaucoma is often treated with prostaglandineye drops. Current treatments are invasive and expensive and burdensomeon the patient and clinic, requiring monthly eye injections (AMD) ordaily eye drop administration.

Ocular gene therapies have the potential to profoundly improve thequality of life in patients with inherited retinal disease. Severalfactors make the eye an ideal organ for gene-replacement therapy. Theeye is accessible, it is a compartmentalized, privileged site. This inturn means that immunologically the eyes are able to tolerate theintroduction of foreign proteins/antigens without eliciting aninflammatory immune response. Clinical trials can take advantage ofcontralateral controls. For this reason, gene therapies for eye diseasesare in development.

In this regard, recombinant adeno-associated virus (rAAV) vectors haveemerged as promising tools for mediating gene therapy for diseases ofthe retina. Effectively controlling gene expression levels followingvector delivery is paramount to the success of potential gene therapies,where uncontrolled over-expression of the therapeutic transgenes canlead to toxicity. Traditionally, inducible promoter systems have beenemployed. Unfortunately, due to the limited coding capacity of AAV, andthe large size of the regulatory elements required to make such systemswork effectively, inclusion of traditional promoters is not feasible.

Riboswitches are a possible alternative. Riboswitches are specificregulatory components of an mRNA molecule that bind and target smalltarget molecules thereby regulating the expression of theriboswitch-containing mRNA's protein product in a cis-fashion. Thus, anmRNA that contains a riboswitch is directly involved in regulating itsown activity, in response to the concentrations of its target molecule.Some riboswitches are “self-targeting”, which means that can regulatetheir own expression. The switch includes an RNA element that can adaptto one of two mutually exclusive secondary structures. One of thesestructures is a signal for gene expression to be “on” and the otherconformation turns the gene “off.”

However, often toxic concentrations of ligand are necessary to “flip theswitch” to turn on or off gene expression. Consequently, a goal ofcurrent research is to improve these regulatory devices towardefficiency, improved regulatory parameters, and clinical applicability.The subject of this invention are riboswitches that can turn on or offgene expression in the retina for use in ocular gene therapy.

SUMMARY OF THE INVENTION

The present invention overcomes the aforementioned drawbacks byproviding constructs, kits and methods of regulating a transgeneexpression using a modified riboswitch which works by a dual mechanismof gene silencing. The invention provides a distinct and improved designover prior riboswitches with its dual mechanism for gene silencing,thereby allowing for better control of expression of the transgene.Novel modified riboswitches are described in more detail herein,including a self-targeting ligand inactivating microRNA (SLIM) switchwhich is an ON-type switch that mediates an increase in gene expressionin the presence of the ligand. Compositions and methods of using thistechnology to treat AMD are provided herein. Specifically, this SLIMswitch can be used to intermittently switch on expression of transgene,for example, a VEGF inhibitor. Further embodiments of SLIM switches thatcan be used to treat glaucoma. Compositions and method of use of thistechnology are contemplated for the treatment of glaucoma, where itwould be beneficial to intermittently turn off, or decrease expressionof the prostaglandin in the eye of a patient.

The foregoing and other aspects and advantages of the invention willappear from the following description. In the description, reference ismade to the accompanying drawings which form a part hereof, and in whichthere is shown by way of illustration a preferred embodiment of theinvention. Such embodiment does not necessarily represent the full scopeof the invention, however, and reference is made therefore to the claimsand herein for interpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a schematic demonstrating the OFF-Switch and ON-Switchriboswitches.

FIG. 2A is a schematic representing the development of a SLIM switch(e.g. switchable miRNA) using a pri-miRNA and an aptamer.

FIG. 2B is a schematic showing the design and functioning of a SLIMswitch with and without ligand.

FIG. 2C is a schematic representing the rAAV vector encoding Aflibercept(a VEGF inhibitor), with the modified switch (smiRNA) and target miRNAsequences (miRT).

FIG. 3 is a schematic of on-type aptamers that can be used in the designof SLIM switches.

FIG. 4 depicts the components of a Theo-SLIM construct.

FIG. 5 is a sequence of an exemplary construct containing Aflibercept (aVEGF inhibitor), Theo-SLIM and three miRNA target sites.

FIG. 6A is fluorescent imaging depicting GFP expression in the retina ofa mouse 4 weeks post infection. C57BL6/J mouse injected intravitreallywith 1.0×10¹⁰ vector genomes (vg) of rAAV2.smCBA-hGFP-3×-L2Bulge9.

FIG. 6B is fluorescent imaging of GFP two hours post-gavage @ 10 mg/kgof theophylline (activating ligand).

FIG. 7 is proposed response of using a SLIM switch (constitutively offtransgene), showing monthly administration of ligand results in a spikein expression level of the transgene (e.g. aflibercept).

FIG. 8A is a schematic of the testing protocol for determining theoptimal copy number of each riboswitch.

FIG. 8B is a schematic of the testing protocol for evaluation of dynamicrange for each riboswitch.

FIG. 9A is a schematic representation of the mRNA of a L2Bulge18tcriboswitch construct.

FIG. 9B is a bar graph depicting the results of different copy numbersof the L2Bulge18tc riboswitch on expression levels of a transgene (GFP).

FIG. 9C is a line graph depicting the dynamic range of a constructcomprising 3 copies of the L2Bulge18tc riboswitch.

FIG. 9D are fluorescent imaging of cells treated with 0-100 μMtetracycline.

FIG. 10A a schematic representation of the mRNA of a constructcontaining a K19 riboswitch.

FIG. 10B is a bar graph depicting the results of different copy numbersof the K19 riboswitch on transgene expression levels.

FIG. 10C is a line graph depicting the dynamic range of the optimal copynumber of K19 riboswitch.

FIG. 10D are fluorescent imaging of cells treated with 0-100 μM oftetracycline which were transduced with the K19 riboswitch construct of10A.

FIG. 11A depicts the mRNA of a construct comprising the L2Bulge9riboswitch.

FIG. 11B is a bar graph depicting the results of different copy numbersof the L2Bulge9 riboswitch on transgene expression levels.

FIG. 11C is a line graph depicting the dynamic range of the optimal copynumber of L2Bulge9 riboswitch.

FIG. 11D are fluorescent imaging of cells treated with 0-100 μM oftheophylline which were transduced with the L2Bulge9 riboswitchconstruct of 11A.

FIG. 12A depicts the mRNA of a construct comprising the tetracyclineSLIM switch (ON-type switch).

FIG. 12B depicts the dynamic range of the tet-SLIM switch of FIG. 12A.

FIG. 12C are fluorescent imaging of cells treated with 0-100 μM oftetracycline which were transduced with the tet-SLIM switch construct of12A.

FIG. 13A depicts the mRNA of a construct comprising the theophyllineSLIM switch (ON-type switch).

FIG. 13B depicts the dynamic range of the theo-SLIM switch of FIG. 13A.

FIG. 13C are fluorescent imaging of cells treated with 0-100 μM oftheophylline which were transduced with the theo-SLIM switch constructof 13A.

FIGS. 14A-14L are representative fluorescein angiography images of CNVlesions. Leakage from CNV lesions was assessed 7 days following laserinjury. Representative FA images taken 5 minutes after fluoresceininjection for mice injected with either (A-C) rAAV2[MAX].smCBA-Eylea,(D-F) rAAV2[MAX].smCBA-Eylea-1×-TC45+standard diet, (G-I)rAAV2[MAX].smCBA-Eylea-1×-TC45+tetracycline diet or PBS (n=16-20 lesionsper group). Images of 3 mice per group with red arrows indicating siteof the laser injury.

FIGS. 15A-15B demonstrate intraocular concentration of Eylea correlatedstrongly with severity of CNV lesions. (A) Distribution of lesionsgraded independently by three blinded scientists. N=16-20 lesions pergroup, p<0.0001, Chi-squared test). (B) Intraocular levels ofnon-complexed Eylea assayed by ELISA. N=5 eyes per group.

FIG. 16 are exemplary sequences of the recombinant VEGF inhibitor cDNA(SEQ ID NO:18), and complete AAV vector comprising SLIM Eylea (SEQ IDNO:19).

FIGS. 17A-17C are schematic representations of 6S-folini acid-responsiveSLIM (A) (SEQ ID NO:21), theophylline-responsive SLIM (B) (SEQ ID NO:26)and tetracycline-responsive SLIM (C) (SEQ ID NO:31).

FIG. 18 are exemplary 6-S-Folinic Acid-responsive miRNA Switches (EyleaCDS target) (SEQ ID NOs:21-25), Theophylline-responsive miRNA switches(Eylea CDS target) (SEQ ID NOs:26-30), tetracycline-responsive miRNAswitches (Eylea CDS target) (SEQ ID NO:31-35), and the soluble fms-liketyrosine kinase 1 (sFLT1) VEGF inhibitor cDNA (SEQ ID NO:36) that can beused with the SLIM switches to treat AMD.

FIG. 19 shows exemplary sequences for 6S-Folinic Acid-responsive miRNASLIM Switches (sFLT1 CDS target) including SEQ ID NOs:37-41,theophylline-responsive miRNA SLIM switches (sFLT1-CDS target) includingSEQ ID NOs:42-46, and tetracycline-responsive miRNA switches (sFLT1-CDStarget) SEQ ID NOs:47-51.

FIG. 20 demonstrates the enzymes of the PGF2α biosynthesis pathway.

FIG. 21 is a depiction of a vector contruct of a vector (e.g. AAVvector) comprising prostaglandin endoperoxide synthase 2 (PTGS2)andPTGFR.

FIG. 22 is a graph depicting the change in intraocular pressure (IOP)from baseline in mice after treatment with an AAV with SLIM riboswitchand administration of the ligand tetracycline.

FIG. 23 are exemplary sequences for use in the present invention,including a codon optimized PTGS2 sequence (SEQ ID NO:52), a codonoptimized PTGFR (SEQ ID NO:53), PTGS2-P2-PTGFR sequence (SEQ ID NO:54),and complete AAV expression cassette for PDG2alpha biosynthesis (SEQ IDNO:58). Also included are additional riboswitches that can be used inthe practice of the present invention (SEQ ID NO:64-67).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides modified riboswitches (e.g., modifiedmiRNA switches) and systems containing said modified riboswitches foruse in the treatment of diseases, specifically eye diseases, and morespecifically AMD and glaucoma. The present invention aims to providenovel systems to regulate gene expression in the eye, using novelmodified microRNA (riboswitches) as a regulatory tool. Such geneticconstructs, and kits or methods utilizing same, may be of use incontrolling gene expression, for example in the production ofrecombinant proteins or transgenes involved in the regulation ofspecific compounds, for example, prostaglandins in the eye.

Such RNA-based systems (riboswitches) offer three distinct advantagesover inducible promoter systems. First, they have a small geneticfootprint (˜100 bp) and so can be easily incorporated within the rAAVvector genome without sacrificing significant amounts of codingcapacity. Second, these devices act in cis, limiting the likelihood ofan immune response as no protein cofactors are required forfunctionality. Last, the aptamer domain of a riboswitch can beengineered to respond to almost any activating ligand, includingproteins, small molecule drugs or ions.

One embodiment of this technology is to modulate expression oftransgenes involved in prostaglandin F2α synthesis in the eye toregulate intraocular pressure in glaucoma patients. A second embodimentis to modulate the expression of an anti-VEGF recombinant fusion proteinin the eye, namely Aflibercept (Eylea) in order to prevent choroidalneovascularization (CNV).

The technology in some embodiments uses constructs including modifiedriboswitches, which are ligand-controlled gene regulatory elements thatallow for either switching on or off a transgene of interest in order toregulate transgene expression. The mechanisms by which riboswitchesfunction include, without limitation, the ability to function as aribozyme and cleave itself if a sufficient concentration of its ligandis present, the ability to fold the mRNA in such a way the ribosomalbinding site is inaccessible and prevents translation from occurring,and/or the ability to affect the splicing of the pre-mRNA molecule.Embodiments comprising the modified riboswitches and constructs orcassettes containing such riboswitches are contemplated herein.

MicroRNAs are a class of non-coding RNAs that play key roles in theregulation of gene expression. Acting at the post-transcriptional level,microRNA (miRNA) genes are transcribed by RNA polymerase II as largeprimary transcripts (pri-mRNA) that are processed by a protein complexcontaining the RNase III enzyme Drosha, to form a precursor microRNA(pre-microRNA). In the present invention, the pri-microRNA have beendesigned to be processed into a pre-mRNA that can act as a silencing RNAto silence gene expression.

Riboswitches are divided into two parts: an aptamer and an expressionplatform. The aptamer is a “sensor” to which a target small moleculeligand binds; the expression platform undergoes structural changes inresponse to the changes in the aptamer sensor. Binding of the aptamerdomain causes conformational changes within the expression platform,this conformational change regulates gene expression by turning the geneoff or on. This is illustrated in FIG. 1. The aptamer is shown in blue;the expression platform, in red; the ligand in green. Often toxicconcentrations of ligand are necessary to “flip the switch”. However,the present invention provides modified riboswitches that can turn on oroff gene expression in the retina for use in ocular gene therapy usingnon-toxic levels of ligand.

A number of exemplary riboswitches are described herein, namely aself-targeting ligand inactivating microRNA (SLIM) embodiments. Aself-targeting ligand inactivating microRNA (SLIM) is an ON-type switchthat mediates an increase in gene expression in the presence of theligand (See, e.g., FIG. 2A and 2B). Compositions and methods of usingthis technology to treat AMD and Glaucoma are provided herein.Specifically, this SLIM system can be used to intermittently switch onexpression of the VEGF inhibitor. Compositions and methods of use ofthis technology are contemplated for the treatment of glaucoma, where itwould be beneficial to intermittently turn off or lower expression ofthe prostaglandin in the eye of a patient are contemplated. Both aredescribed in more detail below.

SLIM: On-Type Modified Riboswitch for Modifying Gene Expression

The present invention in one embodiment provides a self-targeting ligandinactivating microRNA (SLIM) switch which is an ON type riboswitch thatcan be incorporated into an expression construct to regulate expressionof a transgene of interest. The SLIM switch (smiRNA in FIG. 2A and B)requires the basal region of a pri-miRNA to be replaced with an aptamerthat can bind to a target ligand. This sequence is cloned into eitherthe 3′-or 5′ untranslated region of an expression construct. Binding ofthe ligand alters the conformation of the aptamer and pri-miRNA,resulting in non-cleavage of the pri-miRNA by Drosha. A target miRNAsequence is cloned into either the 3′ or 5′ untranslated region whichallows for a second level of regulation, in which the pri-miRNA whencleaved in the absence of ligand is processed into a miRNA that can bindto the target miRNA sequence and prevent transcription. Once the SLIMswitch is incorporated within an expression construct, the transgeneexpression is turned off when ligand is not present, and is turned ononce the ligand binds to the aptamer.

SLIM switches function by regulating gene expression at thepost-transcriptional level. In conditions in which the activating ligandis absent, the pri-miRNA will be cleaved by Drosha from the nascenttranscript. This miRNA will be processed and act as a second mechanismof gene silencing, through binding of the complementary target sites.When the activating ligand is present, gene expression is unaltered andthe gene is expressed.

In one embodiment, an exogenous nucleic acid construct for regulatingexpression of a transgene is provided. The construct encodes (a) atransgene, (b) a smiRNA switch (SLIM switch) located within theuntranslated region of the transgene, wherein the smiRNA switchcomprises an aptamer domain capable of binding to a ligand and apri-miRNA sequence, and (c) at least one miRNA target sequencecomplementary to at least a portion of the pri-miRNA. The miRNA switchregulates expression of the transgene by both (1) regulation of thecleavage of the mRNA (which removes either the poly-A tail or 5′-capdestabilizing the RNA), and (2) regulating cleavage of the pri-miRNAfrom the smiRNA, wherein at least a portion of the cleaved pri-miRNA isprocessed and binds to the at least one miRNA target sequences silencingthe transgene expression.

As discussed above, in the absence of ligand, the pri-miRNA is cleavedfrom the transcript and binds to the at least one miRNA target sequencewhich silences the transgene. This is depicted in FIG. 2B. In someembodiments, the at least one miRNA target sequence complementary to atleast a portion of the pri-miRNA (c) discussed above is encompassed inthe transgene (as depicted in the bottom figure of FIG. 2B). In otherwords, the SLIM switch can be generated toward the transgene itself

In a preferred embodiment, the heterologous self-targeting ligandinactivating microRNA (SLIM) switch comprises (a) a target gene ofinterest, (b) at least one smiRNA switch located within the untranslatedregion of the transgene, wherein the smiRNA switch comprises an aptamerdomain capable of binding to a ligand and a pri-miRNA, wherein thesmiRNA switch regulates expression of the transgene by both (1)regulation of the cleavage of the mRNA of the transgene, and (b)regulating cleavage of the pri-miRNA from the smiRNA, wherein at least aportion of the cleaved pri-miRNA binds to a portion of the transgene (inother words, a portion of the transgene acts as the miRNA targetingsequence) silencing the transgene expression. Suitable riboswitches aredescribed in FIGS. 18 and 19, which also include the portion of theEylea transgene or Flt1 transgene each riboswitch targets (e.g. SLIMswitch for Eylea Target 1 (e.g., SEQ ID NO: 21, or 26 or 31) targets aportion of Eylea found in Eylea target 1 (SEQ ID NO:70), SLIM switch forTarget 2 (e.g. SEQ ID NO:22, 27, 32) target Eylea Target 2 (SEQ IDNO:71) and so forth (e.g. SLIM switch for Eylea 3 targets Eylea Target3, SLIM switch for Eylea 4 targets Eylea Target 4, SLIM switch for EyleaTarget 5 targets Eylea Target 5, SLIM switch for Flt1 Target 1 targetsFlt1 Target 1, etc.). One or more SLIM switches can be combined withinthe exogenous expression constructs for use in the methods describedherein (e.g., SLIM switch for Target 1, Target 2, Target 3, Target 4,and Target 5 of Elyea or Sflt1 can be combined in any combination for aconstruct to target (e.g. SLIM 1 and 2; SLIM 1 and 3; SLIM 1 and 4; SLIM1 and 5; SLIM 2 and 3; SLIM 2 and 4; SLIM 2 and 5; SLIM 3 and 4; SLIM 3and 5; SLIM 4 and 5; SLIM 1, 2, and 3; SLIM 1, 2 and 4; SLIM 1, 2 and 5;SLIM 1, 3, and 4; SLIM 1, 3 and 5; SLIM 1, 4 and 5; SLIM 2, 3 and 4;SLIM 2, 3 and 5; SLIM 2, 4 and 5; SLIM 3, 4 and 5; SLIM 1, 2, 3, and 4;SLIM 1, 2, 3 and 5; SLIM 2, 3, 4 and 5; and SLIM 1, 2, 3, 4 and 5) ofthe SLIM found in FIGS. 18-19.

In one embodiment, an exogenous nucleic acid construct for regulatingexpression of a transgene by modulating the mRNA of the transgene isprovided. The nucleic acid encoding: (a) a target gene of interest, (b)at least one smiRNA switch located within the untranslated region of thetransgene, wherein the smiRNA switch comprises an aptamer domain capableof binding to a ligand and a pri-miRNA, wherein the smiRNA switchregulates expression of the transgene by both (1) regulation of thecleavage of the mRNA of the transgene, and (b) regulating cleavage ofthe pri-miRNA from the smiRNA, wherein at least a portion of the cleavedpri-miRNA binds to a portion of the transgene (as a miRNA targetingsequence) silencing the transgene expression. In a preferred embodiment,the exogenous nucleic acid construct is a AAV viral vector.

In some embodiments, the SLIM switch generated against Eylea or sFlt1are used for the treatment of AMD as shown in the sequences described inFIGS. 18 and 19.

In one embodiment, an exogenous nucleic acid construct encodes: the SLIMswitch, transgene, and at least one target miRNA sequence. Design ofsuch SLIM switches is depicted in FIG. 2A. In one example, a suitableSLIM switch (smiRNA) encodes an aptamer domain and a pri-miRNA. Suitableaptamer domain is adapted from the aptamer domain of an ON-typeriboswitch known in the art, and include but are not limited to, forexample, L2Bulge18tc (for example, but not limited to, SEQ ID NO:12),K19 (For example, SEQ ID NO:15), L2Bulge9 (for example, SEQ ID NO:11),among others. Suitable aptamer domains are depicted in FIG. 3 anddescribed in the specification below.

Suitable SLIM switches include, but are not limited to our developedTheo-SLIM (SEQ ID NO: 1) which is depicted in FIG. 3 (smiRNA), andTet-SLIM (SEQ ID NO:17) as described herein. These SLIM switches havebeen engineered as to not cross-react with any genes in the humangenome, and thus should not cause any off-target effects.

Additional SLIM switches are depicted in FIGS. 18-23.

The term “exogenous” as it refers to nucleic acid is foreign to (notnormally found in nature in) the prokaryotic host cell, or a recombinantnucleic acid that is not normally found in the prokaryotic host cell. Insome embodiments, the exogenous nucleic acid sequence is a heterologoussequence comprising sequences from a number of different sources ororganisms. The term exogenous also encompasses a construct that includesequences from a different species than the species the construct willbe used. For example, a rAAV construct of the present invention willinclude not only endogenous AAV sequences, but exogenous sequences fromhuman or other sources that are not native to the AAV virus.

The nucleic acid constructs provided herein are synthetic engineeredconstructions containing non-naturally occurring sequences.

The term “transgene” and “target gene of interest” are usedinterchangeably to refer to an exogenous gene which is to be expressedin the desired cell by use of the constructs of the present invention.

Further, suitable aptamers can be designed against various ligands andincorporated into the SLIM switches by linking the aptamer to apri-miRNA sequence (See, e.g., FIG. 2A).

In some embodiments, the constructs comprise a transgene and a SLIMswitch. In some of these embodiments, the transgene acts as the miRNAtarget sequence for the SLIM switch. In other embodiments, theconstructs further comprises the SLIM switch which also contain at leastone target miRNA sequence, preferably from about 1-4 target miRNAsequences in the 3′ or 5′ UTR. For example, FIG. 2B (top figure) shows asuitable design of such construct. Suitable ligands are discussed morebelow, and specifically include ligands that are able to cross the bloodretinal barrier, for example, tetracycline, theophylline and guanine.

In some embodiments, the construct comprises at least one target miRNAsequence, alternatively at least two, alternatively at least three,alternatively at least 4 target miRNA sequences. The number of targetmiRNA sequences added may depend on the vector used, for example, in theuse of a rAAV vector, a suitably number of target miRNA include, forexample, from 1-5. Adding more target sites would limit the availablecoding sequences as there is a limit to the size of the rAAV vectors,thus depending on the size and sequence of the transgene may also alterthe number of target miRNA sequences added.

In one embodiment, when the smiRNA is SEQ. ID NO.1, the target miRNAsequence is encoded in SEQ ID NO: 10 (GAGAGAATCTTCTTTCTGTCTATAAAA). Inone suitable embodiment, the construct contains at least three targetmiRNA sequence, for example, as found in SEQ ID NO: 2.

In one embodiment, the construct comprises a suitable transgene in whichthe expression of the transgene can be induced by administration of theligand. For example, as described in more detail below, the transgenemay be a VEGF inhibitor, specifically a VEGF inhibitor which can beinduced to be expressed in the eye for the treatment of AMD.Specifically, a suitable VEGF inhibitor is aflibercept, which is encodedby the cDNA found in SEQ ID NO: 8.

In another embodiment, the aptamer domain and the pri-miRNA are encodedwithin SEQ ID NO:1. In some embodiments, the construct contains othersequences found within constructs, for example, promoters, enhancers,WPRE elements, and the like. One skilled in the art would be able toincorporate other known elements necessary for transgene expression froma nucleic acid construct.

In some embodiments, the construct is an adeno-associated virus (rAAV),a lentivirus, an adnovirus, a plasmid, a herpes simplex virus, abaculovirus, a bacteriophage, among others. Preferably, the construct isan adeno-associated virus. A suitable rAAV construct is demonstrated inFIG. 2 which incorporates the transgene for aflibercept, a Theo-SLIMswitch and 3 miRNA target sequences, for example, the sequence encodedin SEQ ID NO:9 or SEQ ID NO:19.

In some embodiments, the SLIM switch requires the basal region of apri-miRNA to be replaced with an aptamer. This sequence is cloned intoeither the 3′-or 5′ untranslated region of an expression cassette.Additionally, miRNA target sites that are complementary to the sequenceof the mature miRNA can be included in one or multiple copies at eitherthe 5′ or 3′ untranslated region of the cassette.

Self-targeting Ligand Inactivated miRNAs (SLIM) switches function byregulating gene expression at the post-transcriptional level. Inconditions when the activating ligand is absent, the pri-miRNA will becleaved by drosha from the nascent transcript. This miRNA will beprocessed and act as the second mechanism of gene silencing, throughbinding of the complementary target sites. When the activating ligand isprovided, gene expression is unaltered.

Multiple copies of each riboswitch can be included into the3′-untranslated region of the gene of interest. Each riboswitch appearsto have an optimal number of copies as shown in Table 1. Furthermore, insome embodiments, multiple copies of miRNA target sites can be includedin the 3′ or 5′ untranslated region. The synthetic riboswitches comprisean aptamer and an expression platform and rely on changes in theexpression platform activity for regulating gene expression. The miRNAtarget sites are sequences in the therapeutic cassette (construct) thatare recognized by the mature miRNA/siRNA. As discussed more below, themiRNA target sites may be encompassed within the transgene. In otherwords, the cleaved and processed siRNA from the riboswitch binds to thetransgene and inhibits its expression.

Alternatively, the SLIM switch can be generated towards the transgeneitself, with Eylea and sFlt1 for the treatment of AMD, the sequences arefound in FIGS. 16-18, 19 and 23.

In one embodiment, the nucleic acid construct comprises a target gene ofinterest, for example, a VEGF inhibitor (e.g. Eylea (SEQ ID NO:20) orsFLT1 (SEQ ID NO:36) and at least one miRNA SLIM switch located withinthe untranslated region of the target gene (e.g. at least one selectedfrom SEQ ID NO:21-35 or SEQ ID NO:37-51, respectively of the VEGFinhibitors).

In some embodiments, the nucleic acid construct is an rAAV vectorcomprising the Eylea gene (e.g. SEQ ID NO:19). For example, SEQ ID NO:19provides a complete AAV expression vector for the expression of Eyleausing a SLIM switch. Within SEQ ID NO:19, X marks positions in which aSLIM sequence may be inserted. Suitable SLIM sequences that may beinserted can be found in FIG. 18, and include, but are not limited to,6S-Folinic Acid-responsive SLIM (miRNA) switches, including, forexample, SEQ ID NO: 21, 22, 23, 24, and 25 (activated by the ligand6S-Folinic acid), Theophylline-responsive SLIM, for example, SEQ IDNO:26, 27, 28, 29 and 30 (activated by ligand theophylline), orTetracycline-responsive SLIM, for example, SEQ ID NO:31, 32, 33, 34, and35 (activated by ligand tetracycline). In some embodiments, from 1-5SLIM sequences are inserted within the AAV expression vector,alternatively from 1-3 SLIM sequences. For example, SEQ ID NO:19 mayincorporate 1-5 copies of SEQ ID NO:21, alternatively 1-5 copies of SEQID NO:22, alternatively 1-5 copies of SEQ ID NO: 23, alternatively 1-5copies of SEQ ID NO:24, alternatively 1-5 copies of SEQ ID NO:25,alternatively 1-5 copies of SEQ ID NO:26, alternatively 1-5 copies ofSEQ ID NO:27, alternatively 1-5 copies of SEQ ID NO:28, alternatively1-5 copies of SEQ ID NO:29, alternatively 1-5 copies of SEQ ID NO:30,alternatively 1-5 copies of SEQ ID NO:31, alternatively 1-5 copies ofSEQ ID NO:32, alternatively 1-5 copies of SEQ ID NO:33, alternatively1-5 copies of SEQ ID NO:34, alternatively 1-5 copies of SEQ ID NO:35.

In another embodiment, the nucleic acid construct comprises a targetgene of interest, for example, a VEGF inhibitor sFLT1 (SEQ ID NO:36) andat least one smiRNA SLIM switch located within the untranslated regionof the target gene (e.g. at least one selected from SEQ ID NO:37-51).

In some embodiments, the nucleic acid construct comprises the sFLT1 gene(SEQ ID NO:36). sFLT1 may be inserted within a vector, for example arAAV vector, and flanked by X which marks positions in which a SLIMsequence may be inserted. Suitable SLIM sequences that may be insertedcan be found in FIG. 18, and include, but are not limited to, 6S-FolinicAcid-responsive SLIM (miRNA) switches, including, for example, SEQ IDNO: 37, 38, 39, 40, 41 (activated by the ligand 6S-Folinic acid),Theophylline-responsive SLIM, for example, SEQ ID NO:42, 43, 44, 45, 46(activated by ligand theophylline), or Tetracycline-responsive SLIM, forexample, SEQ ID NO:47, 48, 49, 50, and 51 (activated by ligandtetracycline). In some embodiments, from 1-5 SLIM sequences are insertedwithin the construct, alternatively from 1-3 SLIM sequences. Forexample, SEQ ID NO:36 may incorporate as X from 1-5 copies of SEQ IDNO:37, alternatively from 1-5 copies of SEQ ID NO:38, alternatively from1-5 copies of SEQ ID NO:39, alternatively from 1-5 copies of SEQ IDNO:40, alternatively from 1-5 copies of SEQ ID NO:41, alternatively from1-5 copies of SEQ ID NO:42, alternatively from 1-5 copies of SEQ IDNO:43, alternatively from 1-5 copies of SEQ ID NO:44, alternatively from1-5 copies of SEQ ID NO:45, alternatively from 1-5 copies of SEQ IDNO:46, alternatively from 1-5 copies of SEQ ID NO:47, alternatively from1-5 copies of SEQ ID NO:48, alternatively from 1-5 copies of SEQ IDNO:49, alternatively from 1-5 copies of SEQ ID NO:50, or alternativelyfrom 1-5 copies of SEQ ID NO:51. Alternative combinations of SLIMsequences are contemplated (e.g. for example from 1-5 SLIM sequencesselected from SEQ ID NO:36, 37, 38, 39, 40, and 41). Suitable, one wouldpreferably use SLIM sequences activated by the same ligand in theconstruct, for example 1-5 SLIM sequences that are active by thetetracycline ligand.

In another embodiment depicted in FIG. 21, a nucleic acid constructcomprises PTGS2 (SEQ ID NO:52) and PTGFR (SEQ ID NO:53) (depicted incombination in SEQ ID NO:54 as PTGS2-P2A-PTGFR). Suitable nucleic acidconstruct is depicted in FIG. 23 as SEQ ID NO:81, a AAV expressionvector comprising PGF2alpha biosynthesis regulatory genes (PTGS2 (SEQ IDNO:60) and PTGFR (SEQ ID NO:62) including two synthetic riboswitchesTC40 (SEQ ID NO:59) and TC45 (SEQ ID NO:63). Suitable use of suchconstruct is for the treatment of glaucoma. Ligands

The specific ligand to be used depends on the specific application andaptamer incorporated into the SLIM switches. For the treatment of eyediseases and disorders, ligands that are able to cross the blood retinalbarrier are preferred. Suitable aptamers that can cross the bloodretinal barrier include, but are not limited to, for example,tetracycline, theophylline, guanine, galactitol, progesterone, mannitol,estradiol, dopamine, quinidine, urea, digoxin, uracil, verapamil,thiourea, moxifloxacin, thymine, levofloxin, corticosterone,acetazolamide, testosterone, doxycycline, and combinations thereof.

In a preferred embodiment, the ligand is selected from the groupconsisting of tetracycline, theophylline and guanine.

Suitable routes of administration of the ligands are known in the artand include, oral administering, administering via the eye (e.g.,eyedrops) and the like.

Treatment of Eye Diseases

The constructs of the present technology are particularly useful forgene therapy for the treatment of eye diseases. The eye is aparticularly good target for this type of gene therapy for a number ofreasons. The eye is a highly specialized organ which has evolved totransduce light stimuli into electrical signals and to relay thosesignals to the visual cortex. Light sensation and image formation ismediated through the activation of photoreceptor cells located in theoutermost layer of the neurosensory retina, where incident light focusedby the cornea and lens results in the activation of a signaling cascadeand the propagation of an electrical impulse. Despite its complexity theeye has many traits which make it an attractive organ for gene therapy:it is relatively immune privileged, has a small compartment size, iseasily visualized and examined, and readily accessible with minimal riskto patients undergoing surgery. The retina (AMD) or the cornea(Glaucoma) are the primary targets for gene therapy treatments. Vectordelivery is usually achieved through injection of a fluid suspensioncontaining the therapeutic particles into the anatomically constrainedspace adjacent to the target cells. A rAAV.SLIM.αVEGF or rAAV.SLIM.sFLT1vector would be most beneficial if targeted towards cells of the innerretina, including retinal ganglion cells and Muller glia, and as suchwould be administered via intravitreal injection. TherAAV.SLIM.PTGS2-P2-PTGFR vector would be most beneficial if targetedtowards cells of the cornea and anterior chamber, including cornealendothelial cells, and as such would be administered via intracameralinjection. Numerous rAAV clinical trials using rAAV for ocular genetherapy applications have been successfully completed; however, alltrials to date have utilized a sub-retinal delivery approach designed totarget photoreceptors. A current phase I/II clinical trial (NCT01494805)is underway utilizing rAAV-mediated overexpression of soluble fms-liketyrosine kinase-1 (sFLT), a soluble receptor of VEGF, for the treatmentof AMD. Expression of sFLT in this trial is constitutive, and cannot beregulated, unlike the proposed rAAV.SLIM.αVEGF and rAAV.SLIM.sFLT1technology described herein. No rAAV-based gene therapy clinical trialsare underway for the treatment of glaucoma.

Treatment of AMD

The present invention provides methods of treating age-related maculardegeneration by inhibiting, reducing or alleviating at least one symptomof AMD. AMD is a disease involving multiple tissue layers within theeye, including the choroid, retinal pigment epithelium and theneurosensory retina, and occurs in two forms. Dry AMD is anon-proliferative disease state characterized by progressive geographicatrophy of the central retina. In approximately 10-15% of patients thedisease progresses to a wet form, characterized by abnormal growth ofblood vessels from the choroid into the subretinal space. Choroidalneovascularization (CNV) results primarily as a result of increasedintraocular concentrations of VEGF and is the major vision-threateningsymptom of AMD. Administration of anti-VEGF agents is known tosignificantly reduce the incidence of CNV and is the currentgold-standard treatment AMD. The treatment regimen is invasive, however,requiring repetitive (e.g. monthly or bimonthly) intravitreal injectionof purified recombinant anti-VEGF protein.

The proposed rAAV.SLIM.αVEGF technology would act to significantlyreduce the incidence of CNV formation in AMD patients, and consequentlyprevent vision loss. Critically, the inclusion of the SLIM technologywould allow for anti-VEGF expression to be controlled through dosing ofthe activating ligand.

The rAAV.SLIM.αVEGF vector technology of the present invention would actto significantly reduce the incidence of CNV formation in AMD patients,and consequently prevent vision loss. Critically, the inclusion of theSLIM technology would allow for anti-VEGF expression to be controlledthrough dosing of the activating ligand.

The present invention provides methods of treating, reducing,alleviating or inhibiting at least one symptom of AMD comprisingadministering to the eye of a subject a construct comprising a VEGFinhibitor and a SLIM riboswitch as described herein (including, e.g. ,rAAV.SLIM.αVEGF or rAAV.SLIM.sFLT1 vector), and further administering atherapeutically effective amount of the ligand. The SLIM riboswitch candetermine which ligand is used as described in more detail herein.Suitably, the treatment results in the reduction, alleviation orinhibition of one or more symptom of AMD, for example, a reduction orinhibition of the development of CNV.

In one embodiment, the method of treating, inhibiting or reducing atleast one symptom of AMD comprises administering a construct comprisingone or more ON-type riboswitch operably connected to a transgeneencoding a VEGF inhibitor and administering a therapeutically effectiveamount of the ligand. Suitable ON-type riboswitches are known in the artand include, but are not limited to, for example, L2Bulge18tc (SEQ IDNO:12), K19 (SEQ ID NO:15), and L2Bulge 9 (SEQ ID NO:11). Further, theconstruct may encode an optimal copy number of the ON-type riboswitcheswhich can be incorporated into the construct. The optimal copy number ofthe riboswitch is detailed in Table. 1. For example a construct for thetreatment of AMD may comprise from 1-3 L2Bulge18tc riboswitches and aVEGF inhibitor.

TABLE 1 ON-TYPE Switches and SLIM switches Activating Optimal DynamicSEQ Riboswitches Ligand Copy number Range ID NO: L2Bulge18tcTetracycline 3 12.5 4.3-fold 12 K19 Tetracycline 1 39.1 1.5-fold 15L2Bulge9 Theophylline 3 37.9 1.7-fold 11 Theo-SLIM Theophylline 1 7.210.9-fold  1 Tet-SLIM Tetracycline 1 36.4 2.3-fold 17

The SLIM riboswitches can be incorporated into constructs of the presentinvention to alter gene expression of the transgene by administration ofa therapeutically effective amount of the ligand. By “therapeuticallyeffective amount” we mean an amount or dosage of the ligand that is ableto alter the expression level of the transgene product in a cell. Oneskilled in the art will be able to titrate and determine atherapeutically effective amount to produce the proper response andresult in a reduction of the symptoms of the desired disease to betreated. A therapeutically effective amount also maintains that thelevel of ligand is in a non-toxic dose to the subject. Suitably, thedosage may be given daily, weekly or monthly depending on the particularrequirements for expression in the subject.

Treatment of Glaucoma

The present invention provides for the first time higher functioninggenetic switches in a retinal model which can be used to regulatetransgenes within the eye to treat eye diseases. The SLIM switchesdescribed above have the potential to have an increased dynamic rangeover traditional riboswitches and can be used for the treatment,inhibition or amelioration of one or more symptom of glaucoma, includinghigh intraocular pressure. Prostaglandin synthesis will be regulated byorally ingesting the activating ligand. The dose of the activatingligand will be determined by intraocular pressure (IOP) readings of thepatient through the use of a rebound tonometer.

The present disclosure provides methods of treating glaucoma comprisinggene therapy using an adeno-associated virus encoding the constructcomprising a SLIM switch and one or more genes that regulateprostaglandin synthesis as described herein. In another embodiment, thedisclosure provides a method of treating glaucoma comprisingadministering an adeno-associated virus encoding a construct comprisingone or more genes that regulate prostaglandin synthesis and at least oneON-type riboswitch (SLIM). FIGS. 21 and 23 provide one such example ofthe AAV vector encoding genes necessary for regulation of prostaglandinsynthesis and suitable SLIM riboswitches for use in the presentinvention.

Glaucoma is typified by elevated intra-ocular pressure (IOP) leading toprogressive loss of retinal ganglion cells and, ultimately severe visualimpairment. Increased IOP results from an imbalance between theproduction of aqueous humour by the ciliary body in the eye's posteriorchamber and its drainage through the trabecular meshwork in the anteriorchamber. Glaucoma can be categorized based on whether the drainagethrough the trabecular meshwork is completely (closed angle) orpartially (open angle) blocked. Open angel glaucoma is most common andusually presents with no symptoms other than slow progressive visionloss. Closed angle glaucoma is loss common and is considered to be amedical emergency, presenting with acute eye pain, headaches, blurredvision, excessive lacrimation nausea and vomiting.

Due to its chronic nature, the proposed rAAV.SLIM.PTGS2-P2-PTGFR (SEQ IDNO:81) technology would be appropriate for the treatment of glaucoma.

In one embodiment, a method of reducing, inhibiting or ameliorating atleast one symptom of glaucoma is provided. The method comprisesadministering an exogenous nucleic acid construct to the eye of thesubject. In one embodiment, the exogenous nucleic acid construct encodesa transgene that regulates prostaglandin 2α synthesis and at least onesmiRNA riboswitch. In another embodiment, the exogenous nucleic acidconstruct encodes:(i) a transgene that regulates prostaglandin synthesis(e.g., prostaglandin endoperoxide synthase 2 (PTGS2); (ii) a smiRNAswitch located within the untranslated region of the transgene, whereinthe smiRNA switch comprises at least two riboswitches flanking apri-miRNA, each riboswitch comprising an aptamer operably linked to anexpression platform, and(iii) at least one miRNA target sequencecomplementary to at least a portion of the pri-miRNA, wherein thetransgene is incorporated into cells of the subject and express thetransgene, and (b) administering a therapeutically effective amount ofthe ligand that is able to bind to the aptamer in order to regulateexpression of the transgene within the eye to reduce, inhibit orameliorate at least one symptom of glaucoma. In one embodiment, thesymptom is high intraocular pressure.

Suitable ligands include ligands that can cross the blood retinalbarrier, as described herein.

Suitably, the transgene that regulates prostaglandin 2α synthesisincludes, but is not limited to, for example, PTGS2 (SEQ ID NO:52) amongothers. Other suitable transgenes are known in the art.

Kits

This disclosure provides kits. The kits can be suitable for use in themethods described herein. In one aspects, a kit can include a rAAVvector comprising the constructs as described herein, for example, aSLIM switch containing construct. In some aspects, the kit can include aconstruct comprising rAAV vector encoding a VEGF inhibitor as describedherein. In other aspects, the kit can include a construct comprising arAAV vector encoding one or more genes involved in the regulation ofprostaglandin synthesis as described herein. Further, the kits maycomprise one or more doses of the ligand to be administered afterinitial administration of the rAAV vector. Instructions on the timing ofthe dosages and proper administration methods may be provided.

The terms “subject” and “patient” are used interchangeably and refer toany animal (e.g., a mammal), including, but not limited to, humans,non-human primates, rodents, and the like, which is to be the recipientof a particular treatment. Typically, the terms “subject” and “patient”are used interchangeably herein in reference to a human subject.

The term “treating” or “treatment” includes, but is not limited to,reducing, inhibiting or preventing one or more signs or symptomsassociated with the disease or disorder. For example, treating glaucomainclude, for example, reduction in the intraocular eye pressure (e.g.the symptom of glaucoma being treated is high intraocular pressure).

The terms “effective amount” or “therapeutically effective amount” referto an amount sufficient to effect beneficial or desirable biologicaland/or clinical results.

“Expression platform” Within the context of the disclosure, the portionof the modified riboswitch which mediates the effect on the nucleic acidexpression is known as the expression platform. Preferably, theexpression platform is operably linked to the aptamer domain of theriboswitch, preferably structurally linked, most preferably by a nucleicacid linker. Most preferred is that the aptamer domain is linked to theexpression platform by a nucleic acid sequence. Preferably, the stemconfiguration of the portion of the expression platform changesconfiguration upon binding of a ligand, such that a change in theconfiguration of the stem structure results in a corresponding change inthe structure of the expression platform, between a first configurationwhich enhances expression of the nucleic acid sequence and a secondconfiguration which inhibits expression of the nucleic acid sequence.

“Genetic construct” can include nucleic acid sequences that permit it toreplicate in the host cell. Examples include, but are not limited to aplasmid, cosmid, bacteriophage, or virus that carries exogenous DNA intoa cell. A genetic construct can also include additional selectablemarker genes and other genetic elements known in the art. A geneticconstruct can preferably transduce, transform or infect a cell, therebycausing the cell to express the nucleic acids and/or proteins encoded bythe vector.

“Operably linked” a first element is operably linked with a secondelement when the first element is placed in a functional relationshipwith functional relationship with the second element. For instance, aaptamer is operably linked to an expression platform when the binding ofthe aptamer to its ligand causes conformational changes to theexpression platform which alters the function of the expression platform(for example, if the expression platform is a ribozyme, binding of theaptamer to the ligand can activate the ribozyme activity). Generally,operably linked DNA sequences are contiguous and, where necessary tojoin two protein coding regions, in the same reading frame.

It is to be understood that the invention is not limited to theparticular embodiments described. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. The scope of thepresent invention will be limited only by the claims. As used herein,the singular forms “a”, “an”, and “the” include plural embodimentsunless the context clearly dictates otherwise.

It should be apparent to those skilled in the art that many additionalmodifications beside those already described are possible withoutdeparting from the inventive concepts. In interpreting this disclosure,all terms should be interpreted in the broadest possible mannerconsistent with the context. Variations of the term “comprising” shouldbe interpreted as referring to elements, components, or steps in anon-exclusive manner, so the referenced elements, components, or stepsmay be combined with other elements, components, or steps that are notexpressly referenced. Embodiments referenced as “comprising” certainelements are also contemplated as “consisting essentially of” and“consisting of” those elements. In places where ranges of values aregiven, this disclosure explicitly contemplates other combinations of thelower and upper limits of those ranges that are not explicitly recited.For example, recitation of a value between 1 and 10 or between 2 and 9also contemplates a value between 1 and 9 or between 2 and 10. Rangesidentified as being “between” two values are inclusive of the end-pointvalues. For example, recitation of a value between 1 and 10 includes thevalues 1 and 10.

The term “consisting essentially of” and “consisting of” should beinterpreted in line with the MPEP and relevant Federal Circuitinterpretation. The transitional phrase “consisting essentially of”limits the scope of a claim to the specified materials or steps “andthose that do not materially affect the basic and novelcharacteristic(s)” of the claimed invention. “Consisting of” is a closedterm that excludes any element, step or ingredient not specified in theclaim. For example, with regard to sequences “consisting of” refers tothe sequence listed in the SEQ ID NO. and does refer to larger sequencesthat may contain the SEQ ID as a portion thereof.

Aspects of the present disclosure that are described with respect tomethods can be utilized in the context of the compositions of matter orkits discussed in this disclosure. Similarly, aspects of the presentdisclosure that are described with respect to compositions of matter canbe utilized in the context of the methods and kits, and aspects of thepresent disclosure that are described with respect to kits can beutilized in the context of the methods and compositions of matter.

The present invention has been described in terms of one or morepreferred embodiments, and it should be appreciated that manyequivalents, alternatives, variations, and modifications, aside fromthose expressly stated, are possible and within the scope of theinvention.

The invention will be more fully understood upon consideration of thefollowing non-limiting examples.

EXAMPLES Example 1 Ability to Regulate Gene Expression in vivo in theEye

This Example demonstrates that six riboswitches respond to a ligand incell culture and in vivo when deliver to the mouse retina.

Six small (˜100 bp) riboswitches (K19, Tc40×45, GuaM8HDV, L2Bulge18tc,L2Bulge9 and Theo6HDV) responded to a ligand in cell culture, and invivo when delivered to the mouse retina using a rAAV2 vector.

The riboswitches were evaluated in HEK293T cells to determine optimalcopy number (largest dynamic range) and dose-responsiveness to itsactivating ligand using a dual luciferase assay. The ligand used weretetracycline and theophylline.

Cell culture experiments revealed significant changes in fireflyluminescence in response to dosing of the appropriate ligand (p<0.01,One-way ANOVA, N=4 all groups).

Next, the optimal copy number of each riboswitch was cloned into a rAAVGFP reporter cassette and packaged in an AAV2 capsid. EachGFP-riboswitch cassette was injected intravitreally into C57B1/6j micein combination with an AAV2 control vector harboring a non-induciblemCherry reporter gene. Four weeks post-injection, mCherry and GFPfluorescence levels were quantified in vivo using a custom ‘Multiline’confocal scanning laser ophthalmoscope (cSLO). Mice subsequentlyreceived a dose of 1000 mg/kg of its activating ligand (tetracycline,theophylline), and fluorescence levels were quantified 2 and 24 hourspost-gavage.

In vivo results demonstrated dosing mice with the activating ligand ofeach riboswitch could achieve a highly significant change in GFPfluorescence at 2 hours post-gavage compared to pre-treatment levels(p<0.01, paired t test, N=6) (FIG. 6A and 6B). Importantly, GFPfluorescence was recovered to pre-treatment levels 24 hours afterreceiving the activating ligand. This shows that genes can be deliveredand expressed in the retina of the eye.

Example 2 Evaluation of Optimal Copy Number of Riboswitches and DynamicRange Example 2A Testing of Known Riboswitches

This Example demonstrates the optimal number of riboswitches that can beused in a construct and the related dynamic ranges. The protocol forboth assays are outlined in FIG. 8A and 8B. For determining optimal copynumbers, plasmids containing 0-4 copies of each riboswitch were createdwith the transgene as green fluorescence protein (GFP) for readout.HEK293T cells were plated in a 12-well plate and on day 2 transfectedwith 1 μg of each plasmid DNA. On day 3, cells were observed byfluorescent microscopy (excitation 467-498 nm) and fluorescence wasquantified using a plate reader (488 nm excitation 520 nm emission).

For determining dynamic range, constructs containing firefly luciferasecontaining the optimal copy number of the riboswitch were made. HEK293Tcells were plated on day 1, and transfected on day 2 with 1 μg ofplasmid DNA. Cells were treated with 0-100 μM of the ligandcorresponding to the riboswitch and the luminescence was quantified onday 3 using plate reader. Results are shown in Table 2.

As depicted in FIG. 9A, L2Bulge18tc riboswitch was one ON-typeriboswitch tested. The cells were treated with 0 μM, 25 μM, 50 μM, 75μM, and 100 μM of tetracycline (ligand), with the fluorescent microscopyresults shown in FIG. 9D. Fluorescence was quantified and results aredemonstrated in FIG. 9B, demonstrating 3 copies provides optimal dynamicrange, as depicted if FIG. 3C.

K19 riboswitch was also tested (FIG. 10) as described above, and theresults are shown in FIG. 9B-D, demonstrating that 1 copy is the optimalcopy number.

L2Bulge 9 was also tested (using activating ligand Theophylline), andthe optimal copy number was determined to be 3.

Results for a number of ON-type riboswitches are summarized in Table 1above.

Example 2B Testing of New SLIM Switches

Experiments as described in Example 2A were also carried out using thenewly designed Tet-SLIM and Theo-SLIM switches including target miRNAsequence, as depicted in FIG. 12A and 13A, respectively. FIG. 12B and13B show the dynamic range for both switches, while FIG. 12C and 13Cdepict fluorescence as seen in the cells treated with 0-100 μM of theirrespective ligands. Both SLIM riboswitches had an increased dynamicrange over similar riboswitches known in the art.

Example 3 Mouse Study using SLIM Containing Anti-VEGF Compound

Adult (>2 months old) wild-type (e.g. C57B1/6j strain) mice (n=20) willbe purchased from an approved supplier (e.g. Jackson Laboratories) andgroup housed at the Medical College of Wisconsin in standard conditions.After a period of acclimatization, each animal will undergo bilateralintra-vitreal injections. One eye will receive 2 μl sterile buffer(HBSS+0.014% tween-20) containing purified recombinant adeno-associatedvirus (rAAV) packaging the cDNA sequences required for biosynthesis of avascular endothelial growth factor (VEGF) inhibitor (e.g. Aflibercept(Eylea)) under control the SLIM gene-switch (named herein,rAAV.SLIM.αVEGF). The contralateral eye will receive an injection ofbuffer only to control for the effects of the surgical intervention.Four weeks will be allowed for incorporation of the rAAV.SLIM.αVEGFvector into cells of the retina; based on preliminary data we expectganglion cells and Muller glia be effectively transduced. All eyes willbe imaged by fluorescein angiography (FA) using a confocal scanninglaser ophthalmoscope to establish the baseline integrity of the retinaland choroidal blood vessels. Animals will subsequently be assignedrandomly to either the treatment (receives activating ligand) or control(no activating ligand) group. Acute choroidal neovascularization (CNV)will be induced in all eyes by making a small hole in Bruch's membranewith a focused infrared laser beam. The extent of CNV formation will beassessed seven and 14 days thereafter by FA.

It is anticipated that animals of the control group (no ligand=low-levelαVEGF expression) will demonstrate CNV formation in the rAAV.SLIM.αVEGFinjected eyes that is similar in extent to the contralateral bufferinjected eyes. By contrast, it is anticipated that animals of theexperimental group (with ligand=high-level αVEGF expression) willexhibit significantly reduced CNV formation in rAAV.SLIM.αVEGF injectedeyes compared to the contralateral buffer injected eyes. This will serveto demonstrate that rAAV-mediated over-expression of a VEGF inhibitor isan effect method for preventing CNV formation, and that the expressionlevels of the VEGF inhibitor can be modulated (i.e. increased) throughsupplementation of the activating ligand, leading to a reduction in CNVformation. All animals will subsequently be euthanized to allowcollection of the eyes for biochemical analysis, allowing directquantification of aVEGF protein levels within treated (with ligand) andcontrol (no ligand) eyes.

Example 4 Treatment of AMD using rAAV.SLIM.αVEGF Vector

Due to the slowly progressing nature of AMD, a therapeutic window existsbetween initial diagnosis and the onset of severe visual impairment inwhich to intervene. Following diagnosis, patients would receive a singleintravitreal injection of the rAAV.SLIM.αVEGF vector suspended in aphysiologically relevant buffer. The current treatment paradigm for AMDpatients involves monthly or bimonthly intravitreal injections ofanti-VEGF protein; a single-dose administration rAAV.SLIM.αVEGF wouldtherefore represent a significantly less invasive treatment alternative.Intravitreal injections of anti-VEGF protein are currently performedunder local anesthesia as an outpatient procedure; it is anticipatedthat this would also be the case for intravitreal administration of therAAV.SLIM.αVEGF vector. Four to eight weeks would be allowed forincorporation of the rAAV.SLAM.F2α vector into cells of the innerretina. The patient could continue to receive conventional anti-VEGFtherapy during this time. The SLIM technology is an ON-type switch; assuch anti-VEGF protein will not be expressed in the patient's eye untilthe activating ligand is provided. Anti-VEGF protein expression levelsin the patient eye will be regulated through oral administration of theactivating ligand (e.g. in tablet form). The SLIM technology isdose-dependent, allowing the patient/physician to precisely modulateanti-VEGF expression levels within the eye. The retinal cells targetedas part of this procedure do not divide. As a consequence, followingincorporation of the vector into those cells, it is anticipated that therAAV.SLIM.aαEGF vector will persist throughout the patient's lifetime.Anti-VEGF expression can therefore be induced at any time throughout thepatient's lifetime by administration of the activating ligand, negatingthe need for repetitive intra-ocular injections.

Example 5 AAV-Riboswitch for Treating AMD

Current treatment methodologies for treating AMD focus on theadministration soluble receptors or neutralizing antibodies raisedagainst VEGF-A protein in order to inhibit its pro-angiogenic function.Eylea (aflibercept), a recombinant fusion VEGF trap licensed by the Foodand Drug Administration for the treatment of wet AMD in 2011, has beenfound to be highly effective at treating CNV in patients with wet AMD.Although this approach has been largely successful at preventing CNVformation, it requires a monthly high-dose intravitreal injection ofanti-VEGF agents throughout a patient's lifetime, resulting in asubstantial financial and economic burden. Moreover, continuous bolusadministration of both Eylea and Lucentis over multiple years has beenshown to accelerate the rate of retinal and choroidal atrophy. Patientsare also at an increased risk for injection related complications suchas endophthalmitis and cataract formation. In this Example, we combineseveral technologies developed within our laboratory to create aninducible rAAV-based gene therapy approach to treat wet AMD following asingle intravitreal administration.

We assessed whether rAAV-mediated over-expression of Eylea is capable ofpreventing CNV formation following laser injury to Bruch's membrane. Byincorporating a tetracycline-responsive riboswitch (1×TC45) riboswitchin the 3′-UTR of the expression cassette, we were also able to addresswhether modulating the intraocular concentration of Eylea led to analteration in the severity of CNV lesions observed. To this end,age-matched C57BL/6J mice were unilaterally injected with either PBS(n=10), 1.0x10¹⁰ vg of rAAV2/2[MAX].smCBA-Eylea (n=10) or 1.0×10¹⁰ vgrAAV2/2[MAX].smCBA-Eylea-1×-TC45 (n=20). Immediately followinginjections, half of the mice (n=10) injected withrAAV2/2[MAX].smCBA-Eylea-1×-TC45 were placed on diet containing 50 mg/gtetracycline. Six weeks post-injection, CNV formation was initiated byrupturing Bruch's membrane using an infrared laser diode (see methods).Seven days following laser injury, neovascular lesion size and leakagewas assessed via fluorescein angiography using cSLO imaging. Themajority of mice ubiquitously expressing Eylea (smCBA-Eylea) either didnot develop lesions at the site of laser injury, or developed smallgrade 1 or 2A type lesions that leaked minimal fluorescein, even after a5-minute period. (FIG. 14A-C). Mice injected with the ‘OFF-type’smCBA-Eylea-1×-TC45 vector and placed on regular diet also predominantlydeveloped only minor lesions, though a small increase in the number of2B lesions were observed compared to the non-inducible Eylea construct(FIG. 14D-F). Lowering Eylea expression through activation of the TC45riboswitch greatly increased the severity of CNV lesions (FIG. 14G-I) tothe extent that they were similar in extent to PBS-sham injected mice(FIG. 14L-J).

Lesion images were graded by three blinded scientists using the gradingsystem described by Krzystolik et al. (see methods for details).Importantly, grade distribution was significantly different for eachtreatment group Mice receiving a sham injection had the highestincidence of clinically significant ‘Grade 2B’ lesions, while miceubiquitously over-expressing Eylea (smCBA-Eylea) had the lowestincidence of ‘Grade 2B’ lesions.

Notably, downregulating Eylea expression through activation of the TC45riboswitch resulted in a considerable increase in the incidence of‘Grade 2B’ lesions compared to rAAV2[MAX].smCBA-Eylea-1×-TC45 injectedmice receiving standard diet (FIG. 15A). Finally, we determined thelevels of non-complexed Eylea in each sample using an Eylea-specificELISA (Eagle Biosciences). As expected, eyes injected with thenon-inducible construct (smCBA-Eylea) contained the highest levels offree Eylea (438 ng/mL). Moreover, high levels of free Eylea weredetected in animals injected with vector containing the tunableconstruct (395 ng/mL), though levels were lower than animals injectedwith the non-inducible construct. Importantly, tetracycline-mediatedactivation of the TC45 riboswitch resulted in a significant 1.75-folddecrease in non-complexed Eylea (p<0.05). (FIG. 15B) The levels of freeEylea correlated strongly with the occurrence of clinically significantlesions.

Example 6

Self-Targeting Ligand Inactivating miRNAs for AMD Treatment in Humans

A SLIM switch requires the basal region of a pri-miRNA to be replacedwith an aptamer. This sequence is cloned into either the 3′-or 5′untranslated region of an expression cassette. Additionally, miRNAtarget sites that are complementary to the sequence of the mature miRNAcan be included in one or multiple copies at either the 5′ or 3′untranslated region of the cassette.

Self-targeting Ligand Inactivated miRNAs (SLIM) switches function byregulating gene expression at the post-transcriptional level. Inconditions when the activating ligand is absent, the pri-miRNA will becleaved by drosha from the nascent transcript. This miRNA will beprocessed and act as the second mechanism of gene silencing, throughbinding of the complementary target sites. When the activating ligand isprovided, gene expression is unaltered.

Multiple copies of each riboswitch (SLIM) can be included into the3′-untranslated region of the gene of interest. Each riboswitch appearsto have an optimal number of copies as shown in Table 2. Furthermore,multiple copies of miRNA target sites can be included in the 3′ or 5′untranslated region.

Due to the slowly progressing nature of AMD, a therapeutic window existsbetween initial diagnosis and the onset of severe visual impairment inwhich to intervene. Following diagnosis, patients would receive a singleintravitreal injection of the rAAV.SLIM.αVEGF vector suspended in aphysiologically relevant buffer. The current treatment paradigm for AMDpatients involves monthly or bimonthly intravitreal injections ofanti-VEGF protein. Thus, a single-dose administration rAAV.SLIM.αVEGFwould therefore represent a significantly less invasive treatmentalternative. Intravitreal injections of anti-VEGF protein are currentlyperformed under local anaesthesia as an outpatient procedure; it isanticipated that this would also be the case for intravitrealadministration of the rAAV.SLIM.αVEGF vector. The patient could continueto receive conventional anti-VEGF therapy during this time. The SLIMtechnology is an ON-type switch; as such anti-VEGF protein will not beexpressed in the patient's eye until the activating ligand is provided.Anti-VEGF protein expression levels in the patient eye will be regulatedthrough oral administration of the activating ligand (e.g. in tabletform). The SLIM technology is dose-dependent, allowing thepatient/physician to precisely modulate anti-VEGF expression levelswithin the eye. The retinal cells targeted as part of this procedure donot divide. As a consequence, following incorporation of the vector intothose cells, it is anticipated that the rAAV.SLIM.αVEGF vector willpersist throughout the patient's lifetime. Anti-VEGF expression cantherefore be induced at any time throughout the patient's lifetime byadministration of the activating ligand, negating the need forrepetitive intra-ocular injections.

Treating Glaucoma with Vectors Comprising Riboswitches

Primary open angle glaucoma (POAG) is the second leading cause ofirreversible blindness worldwide and is characterized by progressiveloss of retinal ganglion cells and atrophy of the optic nerve, leadingto visual field deficits. The major risk factor for POAG is increasedintraocular pressure (IOP) resulting from decreased aqueous humoroutflow. The gold standard clinical therapy for glaucoma is to reduceIOP through a combination of topical drug administration, and inlate-stage disease, surgery. Unfortunately, due to the largelyasymptomatic (i.e. non-painful) nature of glaucoma and the necessity tomaintain a lifelong daily treatment regimen, patient compliance withdrug therapies is extremely poor (<20%), leading to the development ofsevere sight-threatening complications, even in patients diagnosedearly.

Aav-Riboswitch Experiments—a Model for Treating Glaucoma

Prostaglandin analogs (e.g. Latanprost) are administered clinically asesterified pro-drugs that are absorbed across the corneal epithelium andare hydrolyzed into their active form as they pass through the stromaand corneal endothelium before diffusing into the aqueous humor. Bindingof soluble PGF2α analog to PTGFR triggers up-regulation of matrixmetaloprotinase (MMP) production within the ciliary muscle that promotesremodelling of the extracellular matrix and increased uveoscleralaqueous outflow. Native PGF_(2×) is enzymatically derived fromaracadonic acid, which is present in the cornea at high levels, via amultistep process.

De novo biosynthesis and secretion of PGF_(2×) from the cornea into theanterior chamber promises to be an effective strategy for lowering TOPthat improves upon the current gold-standard clinical treatmentapproach. We have demonstrated that over-expression of prostaglandinendoperoxide synthase 2 (PTGS2), the rate limiting enzyme in PGF_(2×)biosynthesis, and PTGFR, causes a significant decrease in IOP over aperiod of three months. Th cDNA sequences (below) for both enzymes wereincorporated into a AAV expression construct (complete sequence below)incorporating either no riboswitch (non-inducible) or with a 3′ TC40 and5′ TC45 tetracycline inducible riboswitch. Mice received intracamerealinjections of PBS (N=10), rAAV.smCBA-PTGFR.P2A.PTGFR (N=10,non-inducible) or rAAV.smCBA-TC40-PTGFR.P2A.PTGFR-TC45 (inducible). IOPwas measured by rebound tonometry before and 12 weeks followinginjection. Half of the inducible mice were placed on tetracyclinecontaining diet for that period. Mice placed on diet (PGF2alphaExpression OFF) showed no decreased intraocular pressure over the 12week period. Mice on normal diet (PGF2alpha expression ON) showed ahighly significant decrease in IOP, similar to when PGF2alpha isconstitutively expressed (no switch).

SEQUENCE LISTING STATEMENT

The application includes the sequence listing through-out thespecification and in the attached sequence listing statememnt. A fewsequences are listed below, but are not to be considered a fullylisting.

L2bulge9 (Theophylline OFF switch) (SEQ ID NO: 11)CTCGAGGCGATCGCAAACAAACAAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTTGTCCAATACCAGCATCGTCTTGATGCCCTTGGCAGTGGATGGGGACGGAGGACGAAACAGCAAAAAGAAAAATAAAAATTTTTTTTTTAATTAATCTTGGGCCC L2bulgel8tc (Tetracycline ON switch)(SEQ ID NO: 12) CTCGAGGCGATCGCAAACAAACAAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTTGTCCAAAACATACCAGATTTCGATCTGGAGAGGTGAAGAATTCGACCACCTGGACGAGGACGGAGGACGAAACAGCAAAAAGAAAAATAAAAATTAATTAATCTTGGGCCC Theo6HDV (Theophylline OFF switch)(SEQ ID NO: 13) ATGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACCACATACCAGCCGAAAGGCCCTTGGCAGGTGGGCGAATGGGACGCACAAATCTCTCTAGCTTCCCAGAGAGAAGCGAGAGAAAAGTGGCTCTCGuaM8HDV (Guanine OFF switch) (SEQ ID NO: 14)ATGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAATGCTATAATCGCGTGGATATGGCACGCAAGTTTCTACCGGGCACCGTAAATGTCCGACTAGTAGCGAATGGGACGCACAAATCTCTCTAG K19 (Tetracycline ON switch)(SEQ ID NO: 15) CAAACAAACAAAGGCGCGTCCTGGATTCGTGGTAAAACATACCAGATTTCGATCTGGAGAGGTGAAGAATACGACCACCTGTAGTATCCAGCTGATGAGTCCCAAATAGGACGAAACGCGCTAAACAAACAAAC TC40 (Tetracycline OFF switch)(SEQ ID NO: 16) CTGAGGTGCAGGTACATCCAGCTGACGAGTCCCAAATAGGACGAAAGGGAGAGGTGAAGAATACGACCACCTAGGCTCGAAAGAGCCTAAAACATACCTTTCCTGGATTCCACTGCTATCCAC Tet-SLIM (SEQ ID NO: 17)CTAGCACGGGTCCCTAAAACATACCGTGAGCGCGAAAGCGCCCCCATTTTGAGTTAGTGAAGCCACAGATGTAACTCAAAATGGGGGCGCTTTCCCGCCTACGGAGAGGTGAAGAATACGACCACCTAGAAGCTTATTGGTACATGATAACACCCCAAAATCGAAGCACTTCAAAAACACCCCAAAATCGAAGCACTTCAAAAACACCCCAAAATCGAAGCACTTCAAAAACACCCCAAAATCGAAGCACTTCAGTCTCAGGCATCGTACGATGTCGACCTGCAGG

1. An exogenous nucleic acid construct for regulating expression of atransgene by modulating the mRNA of the transgene, the nucleic acidencoding: (a) a target gene of interest, (b) at least one smiRNA switchlocated within the untranslated region of the transgene, wherein the atleast one smiRNA switch comprises an aptamer domain capable of bindingto a ligand and a pri-miRNA. (c) at least one miRNA target sequencecomplementary to at least a portion of the pri-miRNA, wherein the atleast one smiRNA switch regulates expression of the transgene by both(1) regulation of the cleavage of the mRNA of the transgene, and (b)regulating cleavage of the pri-miRNA from the smiRNA, wherein at least aportion of the cleaved pri-miRNA binds to at least one miRNA targetsequences silencing the transgene expression.
 2. The construct of claim1, wherein in the absence of ligand, the pri-miRNA is cleaved from thetranscript and binds to the at least one miRNA target sequence, whereinbinding of the portion of the pri-mRNA to the at least one miRNA targetsequence silences the transgene.
 3. The construct of claim 1 or 2,wherein binding of the ligand to the aptamer domain alters theconformation of the smiRNA, wherein the conformation change inhibits theability of the pri-miRNA to be cleaved from the transcript, and whereinthe transgene is able to be translated.
 4. The construct of any of theproceeding claims, wherein the smiRNA switch is encoded by nucleic acidsequence of SEQ ID NO:1.
 5. The construct of any one of the precedingclaims, wherein the at least one miRNA target sequence is encoded by thenucleic acid sequence (SEQ ID NO: 10) GAGAGAATCTTCTTTCTGTCTATAAAA.


6. The construct of any one of the preceding claims, wherein theconstruct encodes three miRNA target sequences, wherein the at leastthree miRNA target sequences are encoded by SEQ ID NO:
 2. 7. Theconstruct of any one of the preceding claims, wherein the constructcomprises one or more smiRNA switches selected from the group consistingof SEQ ID NOs:21-35 and 37-51.
 8. The construct of any one of thepreceding claims, wherein the smiRNA is located within the 3′ UTR andthe at least one miRNA target sequence is located in the 5′UTR.
 9. Theconstruct of any one of the preceding claims, wherein the constructcomprises at least two miRNA target sequences.
 10. The construct of anyone of the preceding claims, wherein the at least one miRNA targetsequence complementary to at least a portion of the pri-miRNA of (c) isa portion of the transgene within the construct to which at least aportion of the cleaved pri-miRNA binds.
 11. The construct of any of thepreceding claims, wherein the transgene encodes a VEGF inhibitor. 12.The construct of claim 11, wherein the VEGF inhibitor is aflibercept andencoded by SEQ ID NO:
 8. 13. The construct of claim 11, wherein the VEGFinhibitor is sFLT1 encoded by SEQ ID NO:36.
 14. The construct of any ofthe preceding claims, wherein the aptamer binds to a ligand that is ableto cross the blood retinal barrier.
 15. The construct of claim 14,wherein the ligand is selected from the group consisting oftetracycline, theophylline, guanine, galactitol, progesterone, mannitol,estradiol, dopamine, quinidine, urea, digoxin, uracil, verapamil,thiourea, mixiflaxacin, thymine, levofloxin, corticosterone,acetazolamide, testosterone, doxycycline, and combinations thereof. 16.The construct of claim 12 or 13, wherein the ligand is selected from thegroup consisting of tetracycline, theophylline, and guanine.
 17. Theconstruct of claim 1, wherein the transgene comprises a gene involved inprostaglandin synthesis.
 18. The construct of claim 17, wherein thetransgene comprises prostaglandin endoperoxide synthase 2 (PTGS2)encoded by SEQ ID NO:52.
 19. The construct of claim 18 furthercomprising PTGFR encoded by SEQ ID NO:53.
 20. The construct of claim 17or 18 further comprising SEQ ID NO:54.
 21. The construct of any of thepreceding claims, wherein the construct is an adeno-associated virus(AAV) vector.
 22. The construct of claim 21, wherein the sequence is SEQID NO:81.
 23. A method of reducing at least one symptom of age-relatedmacular degeneration in a subject in need thereof, the methodcomprising: (a) administering to the eye of the subject an exogenousnucleic acid construct encoding: (i) an anti-VEG inhibitor; (ii) asmiRNA switch located within the untranslated region, wherein the smiRNAswitch comprises a riboswitch comprising an aptamer domain capable ofbinding to a ligand and a pri-miRNA, and (b) administering atherapeutically effective amount of the ligand to the subject in orderto regulate the expression of the anti-VEGF inhibitor and reduce atleast one symptom of age-related macular degeneration.
 24. The method ofclaim 23, wherein the construct is an adeno-associated virus vector. 25.The method any one of claims 23-24, wherein the transgene isaflibercept.
 26. The method of any one of claims 23-25, wherein theligand is able to cross the blood retinal barrier.
 27. The method ofclaim 26, wherein the ligand is selected from the group consisting oftetracycline, theophylline, guanine, galactitol, progesterone, mannitol,estradiol, dopamine, quinidine, urea, digoxin, uracil, verapamil,thiourea, mixiflxacin, thymine, levofloxin, corticosterone,acetazolamide, testosterone, doxycycline, and combinations thereof. 28.The method of any one of claims 23-27, wherein the construct comprisesone or more SLIM selected from the group consisting of SEQ ID NO:21-35and 37-51.
 29. The method of any one of claims 23-28, wherein theconstruct is administered via intra-cameral injection to the eye. 30.The method of any one of claims 23-29, wherein the ligand isadministered orally or via eyedrops.
 31. The method of any one of claims23-29, wherein the exogenous nucleic acid construct comprises one ormore miRNA target sequences within the untranslated region.
 32. A methodof reducing at least one symptom of glaucoma in a subject in needthereof, the method comprising: (a) administering a exogenous nucleicacid construct to the eye of the subject, the exogenous nucleic acidconstruct encoding: (i) a transgene that regulates prostaglandin 2 alphasynthesis; (ii) a smiRNA switch located within the untranslated regionof the transgene, wherein the smiRNA switch comprises at least tworiboswitches flanking a pri-miRNA, each riboswitch comprising an aptameroperably linked to an expression platform, wherein the transgene isincorporated into cells of the subject and express the transgene, and(b) administering a therapeutically effective amount of the ligand thatis able to bind to the aptamer in order to regulate expression of thetransgene within the eye.
 33. The method of claim 32, wherein the atleast one symptom is a reduction the intraocular pressure within the eyeof the subject.
 34. The method of claim 32 or 33, wherein the constructis administered via intra-cameral injection into the eye.
 35. The methodof any one of claims 32-34, wherein the ligand is able to cross theblood retinal barrier.
 36. The method of claim 35, wherein the ligand isselected from the group consisting of tetracycline, theophylline,guanine, galactitol, progesterone, mannitol, estradiol, dopamine,quinidine, urea, digoxin, uracil, verapamil, thiourea, mixiflxacin,thymine, levofloxin, corticosterone, acetazolamide, testosterone,doxycycline, and combinations thereof.
 37. The method of any one ofclaims 31-36, wherein the exogenous nucleic acid construct furthercomprises (iii) at least one miRNA target sequence complementary to atleast a portion of the pri-miRNA.
 37. The method of any one of claims31-37, wherein the smiRNA switch comprises TC40 (SEQ ID NO:59), TC45(SEQ ID NO:63) or a combination of both smiRNA switches.
 38. Anexogenous nucleic acid construct for regulating expression of atransgene by modulating the mRNA of the transgene, the nucleic acidencoding: (a) a target gene of interest, (b) a smiRNA switch locatedwithin the untranslated region of the transgene, wherein the smiRNAswitch comprises an aptamer domain capable of binding to a ligand and apri-miRNA, wherein the smiRNA switch regulates expression of thetransgene by both (1) regulation of the cleavage of the mRNA of thetransgene, and (b) regulating cleavage of the pri-miRNA from the smiRNA,wherein at least a portion of the cleaved pri-miRNA binds to a portionof the transgene as a miRNA targeting sequence silencing the transgeneexpression.
 39. The construct of claim 38, wherein in the absence ofligand, the pri-miRNA is cleaved from the transcript and binds to themiRNA target sequence, wherein binding of the portion of the pri-mRNA tothe at least one miRNA target sequence silences the transgene.
 40. Theconstruct of claim 38 or 39, wherein binding of the ligand to theaptamer domain alters the conformation of the smiRNA, wherein theconformation change inhibits the ability of the pri-miRNA to be cleavedfrom the transcript, and wherein the transgene is able to be translated.41. The construct of any of the proceeding claims, wherein the smiRNAswitch is encoded by nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:21-35 and 37-51.
 42. The construct of any oneof claims 38-41, wherein the smiRNA is located within the 3′ UTR and theat least one miRNA target sequence is located in the 5′UTR.
 43. Theconstruct of any one of claims 38-42, wherein the construct furthercomprises at least one miRNA target sequences.
 44. The construct of anyone of claims 38-43, wherein the transgene encodes a VEGF inhibitor. 45.The construct of claim 44, wherein the VEGF inhibitor is aflibercept andencoded by SEQ ID NO:
 8. 46. The construct of claim 44, wherein the VEGFinhibitor is sFLT1 encoded by SEQ ID NO:36.
 47. The construct of any oneof claims 38-46, wherein the aptamer binds to a ligand that is able tocross the blood retinal barrier.
 48. The construct of claim 47, whereinthe ligand is selected from the group consisting of tetracycline,theophylline, guanine, galactitol, progesterone, mannitol, estradiol,dopamine, quinidine, urea, digoxin, uracil, verapamil, thiourea,mixiflaxacin, thymine, levofloxin, corticosterone, acetazolamide,testosterone, doxycycline, and combinations thereof.
 49. The constructof 48, wherein the ligand is selected from the group consisting oftetracycline, theophylline, and guanine.
 50. The construct of claim 38,wherein the transgene comprises a gene involved in prostaglandinsynthesis.
 51. The construct of claim 50, wherein the transgenecomprises prostaglandin endoperoxide synthase 2 (PTGS2) encoded by SEQID NO:52 and PTGFR encoded by SEQ ID NO:53.
 52. The construct of any oneof claims 38-51, wherein the construct is an adeno-associated virus(AAV) vector.